We are studying the molecular basis of membrane transport in bacteria using both genetic and biochemical approaches. We are determining the number of components in the high and low affinity transport systems for leucine. The high affinity system requires a periplasmic binding-protein which we have purified and crystallized. This component bears a receptor site for leucine, isoleucine and valine. In collaborative studies the amino acid sequencing of the component has been determined and currently the three-dimensional structure is being determined using x-ray crystallographic techniques. Employing genetic studies we have shown that the high affinity and the low affinity systems for the branched-chain amino acid are separate systems that operate independently. We have found that the regulation of leucine transport activity is very complex and involves such factors as the leucyl tRNA, the tRNA-modifying enzyme and the transcription termination factor, rho. We are identifying transport components other than the binding-proteins using chemical crosslinking reagents and affinity chromatography techniques. We are examining the synthesis and insertion of the leucine-binding protein into the bacterial cell envelope using an in vitro protein synthetic system.